The present invention relates to the use of certain nucleic acid analogues in the field of diagnostics, for instance in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities.
Oligodioxyribonucleotides (oligo-DNA""s) are finding increasing use in diagnostics procedures. They are for instance finding use in testing for the presence of specific micro-organisms or for testing for the presence of generic predispositions, for instance to disease, in forensic science and in microbiology generally. The uses of oligo-DNA""s in this field are of course dependent upon the ability of such oligo-DNA""s to hybridise to complementary nucleic acid sequences. By way of example, labelled oligo-DNA probes are used in hybridisation assays to probe immobilised target DNA""s for the presence of specific sequences. In amplification procedures, the hybridisation property of oligo-DNA""s is utilised to hybridise oligo-DNA primers to template molecules to be amplified.
Oligo-DNA""s as long as 100 base pairs in length are now routinely synthesised using a solid support method and fully automatic synthesis machines are commercially available. Attention has nonetheless been given to the possibility of constructing synthetic DNA-analogues capable of hybridising to natural DNA in a sequence specific manner and yet having chemical properties advantageously distinct from DNA itself. This work has been largely motivated by the possible use of such compounds in xe2x80x9canti-sensexe2x80x9d therapeutics where the use of conventional oligo-DNA""s encounters difficulties because such unmodified oligonucleotides have a short half life in vivo due to the natural presence of nucleases, are difficult and costly to prepare in any quantity and are poor at penetrating cell membranes.
For instance, International (PCT) Patent Application WO86/05518 discloses DNA analogues having a backbone bearing a sequence of ligands, typically nucleotide bases, supposedly capable of sequence specific hybridisation to naturally occurring nucleic acids. A number of different backbone structures are disclosed. No specific exemplification of the provision of such compounds is given and there are no data showing the affinity of the claimed analogues for DNA.
International (PCT) Patent Application WO86/05519 claims diagnostic reagents and systems comprising DNA analogues of the same kind but once again, there is no exemplification.
International (PCT) Patent Application WO89/12060 describes oligonucleotide analogues based on various building blocks from which they are synthesised. Whilst there is exemplification of the building blocks, there is no example of actually preparing an oligonucleotide analogue from them and hence no indication of the performance of the analogues.
Furthermore, it is known to modify the DNA backbone with the aim of increasing resistance to nuclease and generally improving the suitability of the DNA for use in anti-sense therapeutic methods. Other attempts to design DNA analogues are discussed in the introductory portion of WO86/05518 mentioned above.
The universal experience has been that modifications of the backbone of natural DNA lead to a decrease in the stability of the hybrid formed between the modified oligonucleotide and its complementary normal oligonucleotide, assayed by measuring the Tm value. Consequently, the conventional wisdom in this area is that modifications of the backbone always destabilise the hybrid, i.e. result in lower Tm values, and therefore the modification should be as minor as possible in order to obtain hybrids with only a slight decrease in Tm value as the best obtainable result.
The present invention relates to the use in diagnostics or in analysis of nucleic acid analogues of novel structure, preferably having the previously unknown property of forming hybrids with complementary sequence conventional nucleic acids which are more stable in terms of Tm value than would be a similar hybrid formed by a conventional nucleic acid of corresponding sequence and/or exhibiting greater selectively for the complementary sequence compared to sequences involving a degrees of mis-match than would be exhibited by said corresponding conventional nucleic acid of corresponding sequence.
The invention provides a nucleic acid analogue for use in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities, which analogue is
(a) a peptide nucleic acid (PNA) comprising a polyamide backbone bearing a plurality of ligands at respective spaced locations along said backbone, said ligands being each independently naturally occurring nucleobases, non-naturally occurring nucleobases or nucleobase-binding groups, each said ligand being bound directly or indirectly to a nitrogen atom in said backbone, and said ligand bearing nitrogen atoms mainly being separated from one another in said backbone by from 4 to 8 intervening atoms.
(b) a nucleic acid analogue capable of hybridising to a nucleic acid of complementary sequence to form a hybrid which is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding to said analogue and said nucleic acid; or
(c) a nucleic acid analogue capable of hybridising to a double stranded nucleic acid in which one strand has a sequence complementary to said analogue, so as to displace the other strand from said one strand.
The separation of the nitrogen bearing atoms in the backbone of nucleic acid analogues defined in paragraph (a) above (PNA""s) is preferably by five atoms. In nucleic acid analogues having the Formula I (below) this has been found to provide the strongest affinity for DNA. However, it may in some cases be desired to reduce the strength of binding between the PNA""s and DNA by spacing one or more of the ligands by a less than optimal spacing, e.g. by a spacing of more than 5 atoms, e.g. by up to 14 atoms or more.
Preferably not more than 25% of interligand spacings will be 6 atoms or more. More preferably not more than 10 to 15% of interligand spacings will be 6 atoms or more. The aza nitrogen atoms which carry the ligands (directly or via linker groups are not themselves counted in the spacings referred to above.
An alternative or additional method for reducing the strength of DNA binding is to omit certain of the ligands, putting in their place a moiety which contributes little or nothing to the binding of DNA, e.g. hydrogen. Preferably, not more than 25% of the ligand positions will be occupied by non-binding moieties, e.g. not more than 10 to 15%.
Representative ligands include either the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine or thiouracil) or artificial bases (e.g., bromouracil, azaadenines or azaguanines, etc.) attached to a peptide backbone through a suitable linker.
In preferred embodiments, the nucleic acid analogues used in the invention have the general formula (I): 
wherein:
n is at least 2,
each of L1-Ln is independently selected from the group consisting of hydrogen, hydroxy, (C1-C4)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L1-Ln being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
each of A1-An is a single bond, a methylene group or a group of formula (IIa) or (IIb): 
where:
X is O, S, Se, NR3, CH2 or C(CH3)2;
Y is a single bond, O, S or NR4;
each of p and q is an integer from 1 to 5, the sum p+q being not more than 10;
each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and
each R3 and R4 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino;
each of B1-Bn is N or R3N+, where R3 is as defined above;
each of C1-Cn is CR6R7, CHR6CHR7 or CR6R7CH2, where R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, (C2-C6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C1-C6)alkoxy, (C1-C6)alkylthio, NR3R4 and SR5, where R3 and R4 are as defined above, and R5 is hydrogen, (C1-C6)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C1-C6)alkyl, or R6 and R7 taken together complete an alicyclic or heterocyclic system;
each of D1-Dn is CR6R7, CH2CR6R7 or CHR6CHR7, where R6 and R7 are as defined above;
each of G1-Gnxe2x88x921 is 
in either orientation, where R3 is as defined above;
Q is xe2x80x94CO2H, xe2x80x94CONRxe2x80x2Rxe2x80x3, xe2x80x94SO3H or xe2x80x94SO2NRxe2x80x2Rxe2x80x3 or an activated derivative of xe2x80x94CO2H or xe2x80x94SO3H; and
I is xe2x80x94NHRxe2x80x2xe2x80x3Rxe2x80x3xe2x80x3 or xe2x80x94NRxe2x80x2xe2x80x3C(O)Rxe2x80x3xe2x80x3, where Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and Rxe2x80x3xe2x80x3 are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers. Alternatively, C and D may be CHR6(CH2)SCHR7 where S may be from 0 to 2.
Preferred peptide nucleic acids have general formula (III): 
wherein:
each L is independently selected from the group consisting of hydrogen, phenyl, heterocycles, e.g. of one, two or three rings, naturally occurring nucleobases, and non-naturally occurring nucleobases;
each R7xe2x80x2 is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;
n is an integer from 1 to 60;
each of k, l and m is independently zero or an integer from 1 to 5; preferably the sum of k and m is 1 or 2, most preferably 1;
Rh is OH, NH2 or xe2x80x94NHLysNH2; and
Ri is H or COCH3.
Particularly preferred are compounds having formula (III) wherein each L is independently selected from the group consisting of the nucleobases thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U), in particular thymine, and n is an integer from 1 to 30, in particular from 4 to 20. An example of such a compound is provided in FIG. 1, which shows the structural similarity between such compounds and single-stranded DNA.
The peptide nucleic acids of the invention may be synthesized by adaptation of standard peptide synthesis procedures, either in solution or on a solid phase. The synthons used may be specially designed monomer amino acids or their activated derivatives, protected by standard protecting groups. The oligonucleotide analogues also can be synthesized by using the corresponding diacids and diamines.
Thus, the monomer synthons used to produce compounds for use on the invention may be selected from the group consisting of amino acids, diacids and diamines having general formulae: 
wherein L, A, B, C and D are as defined above, except that any amino groups therein may be protected by amino protecting groups; E is COOH, CSOH, SOOH, SO2OH or an activated derivative thereof; and F is NHR3 or NPgR3, where R3 is as defined above and Pg is an amino protecting group.
Preferred monomer synthons are amino acids having formula (VII): 
or amino-protected and/or acid terminal activated derivatives thereof, wherein L is selected from the group consisting of hydrogen, phenyl, heterocycles, naturally occurring nucleobases, non-naturally occurring nucleobases, and protected derivatives thereof; and R7xe2x80x2is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids. Especially preferred are such synthons having formula (4) wherein R7xe2x80x2 is hydrogen and L is selected from the group consisting of the nucleobases thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U) and protected derivatives thereof.
In accordance with the invention there is included the use of nucleic acid analogues as hereinbefore defined in the capture, recognition, detection, identification or quantitation of one or more chemical or micro-biological entities. Usually it is envisaged the entity which is detected in the first instance will be a nucleic acid and said entity will be detected via its characteristic sequences of nucleic acid bases by hybridisation.
Nucleic acid analogues as hereinbefore defined may be used in a method of capturing a nucleic acid comprising contacting under hybridising conditions said nucleic acid with a nucleic acid analogue for use in the invention immobilised to a solid support, which immobilised nucleic acid analogue has a sequence of ligands suitable to hybridise to said nucleic acid or nucleic acid analogue to be captured.
The solid support may take a wide variety of forms as is known in connection with the immobilisation of conventional oligo-nucleotides for use in affinity capture. A solid support may for instance be a plate, a filter, a multi-well plate or a dip stick. It may take the form of individual particles such as beads and such particles may be held in a column through which nucleic acid containing solutions may be run to allow the capture of desired species therefrom.
The captured nucleic acid may be recognised, detected, identified or quantitated by a wide variety of methods. Since after washing the captured nucleic acid may be the only nucleic acid remaining in the system, it may be detected by any reagent system suitable for demonstrating the presence of nucleic acid, whether or not specific for the captured sequence. Thus by way of example if the captured nucleic acid is DNA and is captured in single stranded form by a relatively short PNA, overhanging single stranded DNA may be digested by nuclease and the digestion products may be detected by conventional means. If the DNA is double stranded and the PNA is once again relatively short, that part of the DNA which remains in its original double stranded form (i.e. which is not displaced by the PNA) can be detected by conventional DNA intercalators that do not bind to the PNA-DNA duplex. Antibodies which recognise nucleic acids may be used to detect nucleic acids (RNA, dsDNA or ssDNA) bound to the immobilised nucleic acid analogue.
In the affinity capture of nucleic acid species using conventional oligonucleotides immobilised to a solid support, it is necessary normally to purify the target nucleic acid. Nucleases which may be present in the sample are liable to attack the immobilised nucleic acid. Little specific binding is obtained in practice with much non-specific binding. Furthermore, it is necessary to denature the DNA to single stranded form before the capture can take place.
The nucleic acid analogues of Formula I above are not susceptible to attack by nucleases and typically provide higher levels of specific binding by virtue of their higher affinity for nucleic acid of complementary sequence than is obtained using conventional oligonucleotides as the immobilised species. Furthermore, the nucleic acid analogues used in accordance with the invention are typically capable of hybridising to nucleic acids of complementary sequence without those nucleic acids first being denatured into single stranded form. Once the target nucleic acid has been captured, it may be released from the immobilised nucleic acid analogue by subjecting the immobilised nucleic acid analogue and captured nucleic acid to dehybridising conditions such as heat and dimethyl formamide.
By way of example, the immobilised nucleic acid analogue may comprise sequential ligands such as thymine, hybridisable to poly A tails of mRNA to capture the mRNA.
The invention includes an affinity capture column comprising immobilised nucleic acid analogues as described above.
Thus it can be seen that the present invention also pertains to the advantageous use of PNA molecules in solid-phase biochemistry (see, e.g., xe2x80x9cSolid-Phase Biochemistryxe2x80x94Analytical and Synthetic Aspectsxe2x80x9d, W. H. Scouten, ed., John Wiley and Sons, New York, 1983), notably solid-phase biosystems, especially bioassays or solid-phase techniques which concern diagnostic detection/quantitation or affinity purification of complementary nucleic acids (see, e.g., xe2x80x9cAffinity Chromatographyxe2x80x94A Practical Approachxe2x80x9d, P. D. G. Dean, W. S. Johnson and F. A. Middle, eds., IRL Press Ltd., Oxford 1986; xe2x80x9cNucleic Acid Hybridizationxe2x80x94A Practical Approachxe2x80x9d, B. D. Harnes and S. J. Higgins, IRL Press Ltd., Oxford 1987). Present day methods for performing such bioassays or purification techniques almost exclusively utilize xe2x80x9cnormalxe2x80x9d or slightly modified oligonucleotides either physically adsorbed or bound through a substantially permanent covalent anchoring linkage to beaded solid supports such as cellulose, glass beads, including those with controlled porosity (Mizutani, et al., J. Chromatogr., 1986, 356, 202), xe2x80x9cSephadexxe2x80x9d, xe2x80x9cSepharosexe2x80x9d, agarose, polyacrylamide, porous particulate alumina, hydroxyalkyl methacrylate gels, diol-bonded silica, porous ceramics, or contiguous materials such as filter discs of nylon and nitrocellulose. One example employed the chemical synthesis of oligo-dT on cellulose beads for the affinity isolation of poly A tail containing mRNA (Gilham in xe2x80x9cMethods in Enzymology,xe2x80x9d L. Grossmann and K. Moldave, eds., vol. 21, part D, page 191, Academic Press, New York and London, 1971). All the above-mentioned methods are applicable within the context of the present invention. However, when possible, covalent linkage is preferred over the physical adsorption of the molecules in question, since the latter approach has the disadvantage that some of the immobilized molecules can be washed out (desorbed) during the hybridization or affinity process. There is, thus, little control of the extent to which a species adsorbed on the surface of the support material is lost during the various treatments to which the support is subjected in the course of the bioassay/purification procedure. The severity of this problem will, of course, depend to a large extent on the rate at which equilibrium between adsorbed and xe2x80x9cfreexe2x80x9d species is established. In certain cases it may be virtually impossible to perform a quantitative assay with acceptable accuracy and/or reproducibility. Loss of adsorbed species during treatment of the support with body fluids, aqueous reagents or washing media will, in general, be expected to be most pronounced for species of relatively low molecular weight. In contrast with oligonucleotides, PNA molecules are easier to attach onto solid supports because they contain strong nucleophilic and/or electrophilic centers. In addition, the direct assembly of oligonucleotides onto solid supports suffers from an extremely low loading of the immobilized molecule, mainly due to the low surface capacity of the materials that allow the successful use of the state-of-the-art phosphoramidite chemistry for the construction of oligonucleotides. (Beaucage and Caruthers, Tetrahedron Lett., 1981, 22, 1859; Caruthers, Science, 1985, 232, 281). It also suffers from the fact that by using the alternative phosphite triester method (Letsinger and Mahadevan, J. Am. Chem. Soc. 1976, 98, 3655), which is suited for solid supports with a high surface/loading capacity, only relatively short oligonucleotides can be obtained. As for conventional solid-phase peptide synthesis, however, the latter supports are excellent materials for building up immobilized PNA molecules (the side-chain protecting groups may be removed from the synthesized PNA chain without cleaving the anchoring linkage holding the chain to the solid support). Thus, PNA species benefit from the above-described solid-phase techniques with respect to the much higher (and still sequence-specific) binding affinity for complementary nucleic acids and from the additional unique sequence-specific recognition of (and strong binding to) nucleic acids present in double-stranded structures. They also can be loaded onto solid supports in large amounts, thus further increasing the sensitivity/capacity of the solid-phase technique. Further, certain types of studies concerning the use of PNA in solid-phase biochemistry can be approached, facilitated, or greatly accelerated by use of the recently-reported xe2x80x9clight-directed, spatially addressable, parallel chemical synthesisxe2x80x9d technology (Fodor, et al., Science, 1991, 251, 767), a technique that combines solid-phase chemistry and photolithography to produce thousands of highly diverse, but identifiable, permanently immobilized compounds (such as peptides) in a substantially simultaneous way.
It has been found that PNA""s according to Formula I exhibit a property never before observed which is that such a nucleic acid analogue is capable of hybridising to a conventional nucleic acid presented in double-stranded form and is capable under such conditions of hybridising to the strand which has a sequence complementary to the analogue and of displacing the other strand from the initial nucleic acid duplex. Such recognition can take place to dsDNA sequences 5-60 base pairs long. Sequences between 10 and 20 bases are of interest since this is the range within which unique DNA sequences of prokaryotes and eukaryotes are found. Reagents which recognize 17-18 bases are particular interest since this is the length of unique sequences in the human genome.
It has also been observed that when such a hybridisation reaction is conducted in solution, a second strand of the nucleic acid analogue having the same sequence as the first also hybridises to the nucleic acid strand of complementary sequence so as form a triple helix structure in which two similar strands of PNA are hybridised to a single strand of conventional nucleic acid. It is believed that the first PNA strand hybridises by inter-base hydrogen bonding of the usual kind whilst the second strand of PNA is received in the major groove of the initial duplex by Hoogsteen pairing. Where the PNA is immobilised to a solid support, hybridisation to double stranded nucleic acid with displacement of one strand therefrom is observed but the formation of triple helix structures may be prevented by the immobilisation of the PNA.
The invention includes a nucleic acid analogue as defined above incorporating or conjugated to a detectable label. Generally, all those methods for labelling peptides, DNA and/or RNA which are presently known may in general terms be applied to PNA""s also. Thus, methods of labelling will include the use of radio-isotope labels, enzyme labels, biotin, spin labels, fluorophores, chemiluminence labels, antigen labels or antibody labels.
Labelled PNA""s as described above may be used in methods of recognition, detection or quantitation of target nucleic acids comprising hybridising said target to a labelled nucleic acid analogue as defined above of sufficiently complementary sequence to hybridise therewith under hybridising conditions and detecting or quantitating said label of the nucleic acid analogue so hybridised to said target.
Optionally, the target may be immobilised on a substrate prior to the hybridisation.
In such a method, the target may be immobilised to the substrate by the hybridisation of the first region of the target to a capture nucleic acid or nucleic acid analogue having a sequence sufficiently complementary to said first region to hybridise therewith and which is itself immobilised to said substrate and the labelled nucleic acid analogue may be hybridised to a second region of the target.
The ability of at least preferred nucleic acid analogues according to the invention to hybridise to a double-stranded target nucleic acid and to displace one strand therefrom has been described above. The invention includes a method for displacing one strand from a nucleic acid duplex comprising hybridising to said duplex a nucleic acid analogue as defined above having an affinity for the other strand of said duplex sufficient to be able to displace said one strand therefrom.
The invention includes a method of detecting, identifying or quantitating a double-stranded target nucleic acid comprising hybridising thereto a displacing nucleic acid analogue as defined above capable of displacing one strand from a double-stranded target in which the other strand is of complementary sequence to said displacing nucleic acid analogue, wherein said displacing nucleic acid analogue is of sufficiently complementary sequence to said other strand of said double-stranded target to hybridise thereto so as to displace said one strand of said target in single stranded form, and detecting or quantitating the presence of said one strand after displacement from said double-stranded target.
The displaced strand may be broken down into fragments and the presence of said fragments may be detected. The displaced strand may preferably be broken down by attack by a nuclease. Thus, one may detect the present of a specific double-stranded target nucleic acid sequence by hybridising thereto a complementary PNA to produce strand displacement so as to produce single-stranded DNA in the reaction mixture and digest the single-stranded DNA by the use of a nuclease to produce nucleotides whose presence can be detected as an indicator that specifically the target double-stranded DNA was present initially.
The invention further includes kits for use in diagnostics incorporating at least one nucleic acid analogue as defined above and preferably comprising at least one such nucleic acid analogue which is labelled, e.g. a labelled PNA, and at least one detection reagent for use in detecting said label.
Generally, the nucleic acid analogues will be provided in solution in a hybridisation buffer. Such a kit will generally also include at least one wash buffer solution.
Where the nucleic acid analogue is indirectly labelled, e.g. by biotin, the kit may include a conjugate between an enzyme label and a material such as avidin able to bind to the label of the nucleic acid analogue.
Where the nucleic acid analogue is either directly or indirectly enzyme labelled, the kit may comprise a substrate for the enzyme which is suitable to undergo a monitorable reaction mediated by the enzyme.